The present invention relates to bioconjugates and the delivery of bioactive agents which are preferably targeted for site-specific release in cells, tissues or organs. More particularly, this invention relates to bioconjugates which comprise a bioactive agent and an organocobalt complex. The bioactive agent is covalently bonded directly or indirectly to the cobalt atom of the organocobalt complex. The bioactive agent is released from the bioconjugate by the cleavage of the covalent bond between the bioactive agent and the cobalt atom in the organocobalt complex. The cleavage may occur as a result of normal displacement by cellular nucleophiles or enzymatic action, but is preferably caused to occur selectively at a predetermined release site by application of an external signal. The external signal may be light or photoexcitation, i.e. photolysis, or it may be ultrasound, i.e. sonolysis. Further, if the photolysis takes place in the presence of a magnetic field surrounding the release site, the release of the bioactive agent into surrounding healthy tissue is minimized.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
The focus of a substantial body of research has been the development of a system whereby a pharmaceutical agent can be selectively delivered to a desired anatomic location; namely the site in need of treatment. In spite of the great progress which has been achieved in this regard, many pharmaceutical delivery systems for the treatment of various diseases or health risks, e.g., the treatment of cancer, impart substantial risk to the patient. With respect to the treatment of cancer, drugs which are effective in attacking malignant cells to destroy them, or at least limit their proliferation, have a tendency to attack benign cells also. Therefore, it is highly desirable to limit the location of their action to that of the malignancy, and to ensure that at any particular time effective, but not excessive, amounts of such drugs are used.
Although it is desired to concentrate a cytotoxic agent at a targeted site, current cancer treatment protocols for administering these cytotoxic agents typically call for repeated intravenous dosing, with careful monitoring of the patient. The drugs are often used in combination to exert a multi-faceted assault on neoplastic cells. The dose is selected to be just below the amount that will produce acute (and sometimes chronic) toxicity that can lead to life-threatening cardiomyopathy, myelotoxicity, hepatic toxicity, or renal toxicity. Alopecia (hair loss), mucositis, stomatitis, and nausea are other common, but generally not life-threatening, side effects at these doses. Since many of these compounds are potent vesicants, tissue necrosis will occur if localized extravasation (loss of the drug from blood to the surrounding tissue) occurs. These effects occur since the blood generally attains a specified concentration of that drug before becoming effective. Because the blood is transported throughout the body of the host being treated, so is the pharmaceutical agent. Following this technique provides an even distribution of the drug throughout the body, rather than concentrating it at the treatment site. Moreover, such systemic treatment methods expose the healthy cells to the cytotoxic agent concurrent with the treatment of the unhealthy or diseased cells besides limiting the concentration of the drug at the site where it is most needed.
Previous attempts to administer such drugs by direct injection into the location of the organ having the malignancy are only partially effective, because of migration of the drug from that location and as a result of extensive tissue necrosis from extravasation. Such dispersion cannot be totally prevented, with the result that excessive quantities of drug need to be administered to attain a desired result. Although careful clinical monitoring may prevent extensive damage or loss of viable tissue, the providing of a pharmaceutical agent-carrier system which is actively transported through standard biological systems to the treatment site prior to activation of the pharmaceutical agent would be highly desirable not only in optimizing utilization of the drug but also in the reduction of side effects and/or the minimization of the destruction of healthy cells. The direct injection of cytotoxic agents into solid tumors of the breast, bladder, prostate and lung using conventional cytotoxic chemotherapeutic agents as adjuvants to surgery and/or radiotherapy has been of limited success in prolonging the lives of patients. This is partially due to the dose limitations imposed by the acute and chronic toxicity to tissues or organ systems beyond those that are targeted.
As it relates to the administration of cytotoxic or antineoplastic drugs, the effective resolution of concerns relating to modes of administration, to the limitation of dosage size and frequency of administration, and to side effects would certainly be of benefit to the treatment of cancer.
Oligonucleotides that specifically interfere with gene expression at the transcriptional or translational levels have the potential to be used as therapeutic agents to control the synthesis of deleterious proteins associated with viral, neoplastic or other diseases. It is possible to select single-stranded oligonucleotides that recognize and bind to the major groove of a stretch of double-stranded DNA in a sequence-specific manner to form a triple helix (Le Doan et al., 1987; Moser and Dervan, 1987). Triple helix-forming oligonucleotides targeted to the promoter region of certain genes have been used to physically block RNA synthesis in cell-free transcription assays (Cooney et al., 1988; Postel et al., 1992; Skoog et al., 1993; Rando et al., 1994). Similarly, in vitro translation assays have been used to demonstrate that antisense oligonucleotides can bind mRNA targets and prevent protein synthesis (Uhlmann and Peyman, 1990; Cohen and Hogan, 1994).
Although antisense oligonucleotides have shown great efficacy in the selective inhibition of gene expression (Stein and Cohen, 1988; Szczylik et al., 1991; Gray et al., 1993), the therapeutic applications of such antisense oligonucleotides are currently limited by their low physiological stability, slow cellular uptake, and lack of tissue specificity. The instability obstacles have been largely overcome by use of backbone-modified oligonucleotides that are more resistant to nucleases. Methylphosphonates, protein-nucleic acid conjugates, and phosphorothioates all appear to resist enzymatic digestion better than the corresponding natural oligonucleotides (Chang and Miller, 1991; Wickstrom et al., 1992; Letsinger, 1993; Zon, 1993).
Problems with cellular uptake of antisense oligonucleotides have been more difficult to solve. Endogenous uptake pathways that rely on pinocytosis and related processes generally have insufficient capacity to deliver the quantities of antisense of oligonucleotides required to suppress gene expression (Vlassov et al., 1994). Hydrophobic modifications have also been undertaken to improve membrane permeability, but such derivatization strategies are most useful only for short oligonucleotides (Vlassov et al., 1994). Although complexes of antisense constructs with cationic liposomes or immunoliposomes (Gao and Huang, 1991; Bennett et al., 1992, Ma and Wei, 1996) and polylsine (Trubetskoy et al., 1992; Bunnell et al., 1992) have significantly enhanced intracellular delivery, they have simultaneously introduced new disadvantages of their own. Thus, both methods exhibit some carrier cytotoxicity, and like other protocols, neither strategy allows for any tissue or cell targeting. In short, intracellular delivery and tissue specificity remain major obstacles to the implementaiton of antisense drugs in the treatment of human disorders.
Other techniques for the delivery of oligonucleotides to cells include the use of: (a) folate-PEG-liposome constructs for the delivery of antisense DNA against growth factor receptor (Wang et al., 1995); (b) folic acid-polylysine constructs for the delivery of c-myc antisense DNA (Ginobbi et al., (1997); (c) tris(N-acetylgalactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl)-glutamate (YEE(GalNAcAH)3) linked to polylysine for the delivery of DNA to cells via the asialoglycoprotein receptor (Merwin et al., 1994); and (d) water-soluble block polycations (Kabanov et al., 1995).
It has been known for some time that a pharmaceutically active agent can be attached to a carrier or molecule. The term xe2x80x9cprodrugxe2x80x9d is often associated with such systems wherein the active agent is bonded to another molecule for purposes of administration. The drug is usually inactive in the prodrug state and the bond is later cleaved releasing the drug at a site where it can be effective. However, such systems are not as useful as might be desired for various reasons, site specificity being one. Also, the release of the drug from its carrier requires the presence of some agent or event to separate the active drug from its carrier or molecule and, as such, may rely on factors such as the presence of a specific enzyme, pH conditions, time release and the like, which may be variable from host to host and which may not be effectively implemented.
For example, transmembrane transport of nutrient molecules is a critical cellular function. Because practitioners have recognized the importance of transmembrane transport to many areas of medical and biological science, including drug therapy, peptide therapy and gene transfer, there have been significant research efforts directed to the understanding and application of such processes. Thus, for example, transmembrane delivery of nucleic acids has been encouraged through the use of protein carriers, antibody carriers, liposomal delivery systems, electroporation, direct injection, cell fusion, vital carriers, osmotic shock, and calcium-phosphate mediated transformation. However, many of those techniques are limited both by the types of cells in which transmembrane transport is enabled and by the conditions of use for successful transmembrane transport of exogenous molecular species. Further, many of these known techniques are limited in the type and size of exogenous molecule that can be transported across a membrane without loss of bioactivity.
One method for transmembrane delivery of exogenous molecules having a wide applicability is based on the mechanism of receptor-mediated endocytotic activity. Unlike many other methods, receptor-mediated endocytotic activity can be used successfully both in vivo and in vitro. Receptor-mediated endocytosis involves the movement of ligands bound to membrane receptors into the interior of an area bounded by the membrane through invagination of the membrane. The process is initiated or activated by the binding of a receptor-specific ligand to the receptor. Many receptor-mediated endocytotic systems have been characterized, including those recognizing galactose, mannose, mannose 6-phosphate, transferrin, asialoglycoprotein, transcobalamin (Vitamin B12), xcex1-2-macroglobulins, insulin, and other peptide growth factors such as epidermal growth factor (EGF).
Receptor-mediated endocytotic activity has been utilized for delivering exogenous molecules such as proteins and nucleic acids to cells. Generally, a specified ligand is chemically conjugated by covalent, ionic or hydrogen bonding to an exogenous molecule of interest (i.e. the exogenous compound), forming a conjugate molecule having a moiety (the ligand portion) that is still recognized in the conjugate by a target receptor. Using this technique, the phototoxic agent psoralen has been conjugated to insulin and internalized by the insulin receptor endocytotic pathway (Gasparro, 1986); the hepatocyte-specific receptor for galactose terminal asialoglycoproteins has been utilized for the hepatocyte-specific transmembrane delivery of asialoorosomucoid-poly-L-lysine non-covalently complexed to a DNA plasmid (Wu, 1987); the cell receptor for epidermal growth factor has been utilized to deliver polynucleotides covalently linked to EGF to the cell interior (Myers, 1988); the intestinally situated cellular receptor for the organometallic Vitamin B12-intrinsic factor complex has been used to mediate delivery to the circulatory system of a vertebrate host a drug, hormone, bioactive peptide or immunogen complexed with Vitamin B12 and delivered to the intestine through oral administration (Russell-Jones et al., 1995); the mannose-6-phosphate receptor has been used to deliver low density lipoproteins to cells (Murray and Neville, 1980); the cholera toxin binding subunit receptor has been used to deliver insulin to cells lacking insulin receptors (Roth and Maddox, 1983); the human chorionic gonadotropin receptor has been employed to deliver a ricin a-chain coupled to HCG to cells with the appropriate HCG receptor in order to kill the cells (Oeltmann and Heath, 1979); the transferrin receptor has been used to deliver mitomycin C to sarcoma cells (Tanaka et al., 1996) or to deliver doxorubicin to multidrug-resistant cells (Fritzer et al., 1996);the biotin receptor has been employed to deliver hypoxanthine-guanine phosphoribosyl transferase (HGPRT) by biotinylating the HGPRT to restore growth to HGPRT deficient cells (Low et al., 1995); and the folic acid receptor has been used to deliver antisense DNA to src-transformed fibroblast cells (Low et al., 1995).
Russell-Jones et al. (1995), describes a system which involves the formation of a covalent bond between the pharmaceutical agent one wishes to deliver and a modified Vitamin B12 to form a conjugate molecule. The conjugate is orally administered and is then transported from the intestinal lumen to the circulation. Importantly, the pharmaceutical agent and the vitamin are bound through an amide linkage which is prone to acid hydrolysis. Russell-Jones et al. found that many biologically active pharmaceutical agents can be bound to B12 for facilitating the introduction of the drug into the blood stream through oral administration. Importantly, no method was provided whereby the drug-B12 bond could be selectively cleaved, nor could location of the active pharmaceutical agent be controlled once activated. Instead, Russell-Jones et al. relied on biochemical degradation of the drug-B12 bond to release the drug in its active form. Importantly, under this method the drug could be released in its active form anywhere within the circulation system, diminishing the importance of the active transport of B12 into cancer tissue. Moreover, the conjugates formed under this method require the modification of the structure of the corrin ring of the B12 molecule, which modification can have serious effects on receptor interactions.
Thus, there exists a need for a drug delivery system which can be utilized for the delivery of bioactive agents, including pharmaceuticals, peptides and oligonucleotides. There is also a need for a drug delivery system which can be used for site-specific release of the bioactive agent in the cells, tissues, or organs in which a therapeutical effect is desired to be effected.
The present invention relates to bioconjugates and the delivery of bioactive agents which are preferably targeted for site specific release in cells, tissues or organs. More particularly, this invention relates to bioconjugates which comprise a bioactive agent and an organocobalt complex. The bioactive agent is covalently bonded directly or indirectly to the cobalt atom of the organocobalt complex. The bioactive agent is released from the bioconjugate by the cleavage of the covalent bond between the bioactive agent and the cobalt atom in the organocobalt complex, as described herein.
The bioactive agent is any agent which is desired to be delivered to cells, tissues or organs for nutrient or therapeutic effects. In accordance with the present invention, bioactive agents include, but are not limited to, nutrients, pharmaceuticals, drugs, peptides and oligonucleotides.
The organocobalt complex is any organic complex containing a cobalt atom having bound thereto 4-5 nitrogen and/or chalcogens such as oxygen, sulfur, etc., as part of a multiple unsaturated heterocyclic ring system. In accordance with the present invention, suitable organocobalt complexes include, but are not limited to, cobalamin, Co[SALEN], organo-(pyridine)bis(dimethylglyoximato)cobalt, corrinoids, derivatives thereof and analogues thereof. The organocobalt complexes may be unsubstituted or substituted with conventional organic functional groups which will not alter the basic nature of the organocobalt complex. The basic nature of the organocobalt complex is to directly or indirectly bind the bioactive agent covalently to the cobalt such that the cobalt-bioactive agent bond is readily cleavable as described herein. The organocobalt complex may also be covalently bound directly or indirectly to a targeting molecule. The targeting molecule is a molecule for which the desired cell, tissue or organ has a requirement or a receptor, as described herein.
The bioconjugate according to the present invention is administered to a subject in need of therapeutic treatment. The bioconjugate concentrates in a targeted cell, tissue or organ site as a result of the organocobalt complex. As an example, a bioconjugate containing a chemotherapeutic is administered to a patient and the bioconjugate concentrates in neoplastic cells where the active chemotherapeutic is released from the bioconjugate by cleavage. Similarly, other pharmaceuticals, drugs, peptides or oligonucleotides are administered to a subject as part of the bioconjugate which is concentrated in the desired cells, tissues or organs. The pharmaceuticals, drugs, peptides or oligonucleotides are released by cleavage. In one embodiment, the cleavage may occur as a result of normal displacement by cellular nucleophiles or enzymatic aciton. In a second embodiment, the cleavage is caused to occur selectively at the release site by an external signal. The external signal may be light or photoexcitation, i.e. photolysis, or it may be ultrasound, i.e. sonolysis. Further, if the photolysis takes place in the presence of a magnetic field surrounding the release site, the release of the drug, such as a cytotoxic agent, into surrounding healthy tissue can be minimized.